# US 7,745,161 B2 · Amplification of enzymatic reactions for use with an enthalpy array > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 7,745,161 B2 - Document ID: US7745161B2 - Status: Granted - Kind: B2 - Application number: 10/740,776 - Filed: 2003-12-19 - Published: 2005-06-23 - Granted: 2010-06-29 - Earliest claimed: 2003-12-19 - Assignees: Palo Alto Research Center Incorporated; The Scripps Research Institute - Inventors: Francisco E. Torres; Richard H. Bruce; James R. Williamson; Peter Kuhn; Ray Stevens - Web page: https://patentgrep.com/patent/US7745161B2 ## CPC - G01N 25/4846: {for a motionless, e.g. solid sample} - G01N 25/4866: {by using a differential method} - G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q) ## Abstract Provided is a method of detecting characteristics of a reaction of interest, including instituting the reaction of interest, obtaining an amplified heat related to the reaction of interest, measuring the amplified heat, and determining the characteristics of the reaction of interest, using the signal obtained in the step of measuring. ## Claims ### Claim 1 A method of detecting characteristics of an enzymatic reaction of interest comprising: initiating a primary enzymatic reaction wherein a product and a first amount of heat are generated in a volume of approximately 30 microliters or less; activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein the product of the primary enzymatic reaction is converted to a species that activates the amplifying enzyme, wherein the species is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme; measuring the amplified amount of heat; and determining, the characteristics of the reaction of interest using the measured amplified heat. ### Claim 2 (depends on 1) The method according to claim 1, wherein the volume is less than 1 microliter. ### Claim 3 (depends on 1) The method according to claim 1, wherein the volume is less than 300 nanoliters. ### Claim 4 (depends on 1) The method according to claim 1, wherein the amplifying enzyme is an ATPase. ### Claim 5 (depends on 1) The method according to claim 1, wherein the amplifying enzyme is activated as a result of covalent modification by a species converted from a product of the primary enzymatic reaction. ### Claim 6 (depends on 5) The method according to claim 5, wherein the activating enzyme is a kinase and the covalent modification comprises phosphorylation of the activating kinase. ### Claim 7 (depends on 1) The method according to claim 1, wherein a succession of enzymatic reaction cascades are used for generation of the amplified amount of heat in a plurality of reactions. ### Claim 8 (depends on 1) The method according to claim 1, wherein the amplifying enzyme is selected from the group consisting of phosphorylases, phosphofructokinases, and isocitrate dehydrogenases. ### Claim 9 (depends on 1) The method of claim 1 wherein the second enzymatic reaction is initiated by a substrate of the amplifying enzyme that was converted from a specie of a product of the primary reaction. ### Claim 10 (depends on 9) The method of claim 9, wherein the specie converted from a product of the primary reaction is ribose-5-phosphate. ### Claim 11 (depends on 9) The method according to claim 9, wherein the amplifying enzyme is activated by a species converted from ADP. ### Claim 12 A method for monitoring a primary enzymatic reaction comprising: conducting a primary enzymatic reaction wherein a product and a first amount of heat are produced in a volume of 30 microliters or less; producing a detectable amplified amount of heat by activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein the product of the primary enzymatic reaction is converted to a specie that activates the amplifying enzyme wherein the specie is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme; and detecting the amplified amount of heat. ### Claim 13 (depends on 12) The method according to claim 12, wherein the volume is less than 1 microliter. ### Claim 14 (depends on 12) The method according to claim 12, wherein the volume is less than 300 nanoliters. ### Claim 15 (depends on 12) The method of claim 12, wherein the amplifying enzyme is activated as a result of covalent modification by a specie converted from a product of the primary reaction. ### Claim 16 (depends on 15) The method of claim 15, wherein the activating enzyme is a kinase and the covalent modification comprises phosphorylation of the activating kinase. ### Claim 17 (depends on 12) The method according to claim 12, wherein the amplifying enzyme is selected from the group consisting of phosphorylases, phosphofructokinases and isocitrate dehydrogenase. ### Claim 18 (depends on 12) The method according to claim 12, wherein the second enzymatic reaction further comprises a positive feedback loop. ### Claim 19 (depends on 12) The method according to claim 12, wherein the second enzymatic reaction is initiated by a substrate of the amplifying enzyme that was converted from a specie of a product of the primary reaction. ### Claim 20 (depends on 19) The method of claim 19, wherein the specie converted from a product of the primary reaction is ribose-5-phosphate. ### Claim 21 A method for monitoring ATP kinase enzymatic reactions, the method comprising: conducting an ATP kinase enzymatic reaction, the ATP kinase reaction including reacting an ATP kinase with one or more substrates to produce one or more ATP kinase-specific products, the ATP kinase reaction of interest generating a first amount of heat, wherein the ATP kinase and one or more substrates have a combined volume of less than 30 microliters; and activating an amplifying enzyme that initiates a second enzymatic reaction that produces a detectable amplified amount of heat wherein a product of the ATP kinase enzymatic reaction is converted to a species that activates the amplifying enzyme, wherein the species is selected from the group consisting of a substrate for the amplifying enzyme and an agent that causes a covalent modification of the amplifying enzyme; and detecting the heat generated. ### Claim 22 (depends on 21) The method according to claim 21, wherein the ATP kinase is a hexokinase. ### Claim 23 (depends on 22) The method according to claim 22, wherein the substrate is glucose. ### Claim 24 (depends on 22) The method according to claim 22, further comprising a positive feedback loop. ### Claim 25 (depends on 23) The method according to claim 23, further comprising a positive feedback loop. ### Claim 26 (depends on 21) The method according to claim 21, wherein the volume is less than 1 microliter. ### Claim 27 (depends on 21) The method according to claim 21, wherein the volume is less than 300 nanoliters.